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Intersectional monosynaptic tracing for dissecting subtype-specific organization of GABAergic interneuron inputs

Abstract

Functionally and anatomically distinct cortical substructures, such as areas or layers, contain different principal neuron (PN) subtypes that generate output signals representing particular information. Various types of cortical inhibitory interneurons (INs) differentially but coordinately regulate PN activity. Despite a potential determinant for functional specialization of PN subtypes, the spatial organization of IN subtypes that innervate defined PN subtypes remains unknown. Here we develop a genetic strategy combining a recombinase-based intersectional labeling method and rabies viral monosynaptic tracing, which enables subtype-specific visualization of cortical IN ensembles sending inputs to defined PN subtypes. Our approach reveals not only cardinal but also underrepresented connections between broad, non-overlapping IN subtypes and PNs. Furthermore, we demonstrate that distinct PN subtypes defined by areal or laminar positions display different organization of input IN subtypes. Our genetic strategy will facilitate understanding of the wiring and developmental principles of cortical inhibitory circuits at unparalleled levels.

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The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank I. Wickersham for providing us with cell lines necessary for rabies virus production, E. Gomez and S. Laborde for spatial binning analysis, and Taniguchi lab members for careful reading of the manuscript and comments. Chi-square statistical analysis was performed by G. Crynen of The Scripps Research Institute, Florida Campus. This work was supported by Max Planck Florida Institute for Neuroscience (to H.-B.K. and H.T.), the National Institutes of Health Grants MH107460 (to H.-B.K.), DP1MH119428 (to H.-B.K.), MH115917 (to H.T.), and a grant from Japan Science and Technology Agency (PRESTO) (to H.T.).

Author information

H.T. conceived and supervised the project. H.T. and M.J.Y. designed experiments and interpreted results. F.O and E.M.C rescued RVs from DNA plasmids. M.J.Y. and E.W. pseudotyped RVs with EnvA and generated plasmids. M.J.Y. and E.W. conducted experiments except for electrophysiology. Y.H. helped with E12.5 IUE experiments and FISH experiments. J.H.H. performed all electrophysiology experiments and data analyses. H.Z. provided Ai65 mice before publication. M.J.Y. carried out quantitative analyses. H.-B.K. provided comments and edited the manuscript. H.T. and M.J.Y. wrote the manuscript.

Competing Interests

The authors declare no competing interests.

Correspondence to Hiroki Taniguchi.

Integrated supplementary information

  1. Supplementary Figure. 1 Mono-trans-synaptic spread of RV-Flp-CFP viruses depends on expression of RG in starter PNs (Related to Figs. 1 and 2).

    (a) Experimental schema for monosynaptic tracing of input neurons sending direct inputs to supragranular PNs. (b) Confocal projection image merging H2BYFP (yellow) and CFP (cyan). CFP + /H2BYFP + and CFP + /H2BYFP- represent starter PNs, and general input neurons respectively of animals prepared as shown in a. Scale bar, 500 μm. (c,d) Confocal projection images merging DAPI (blue) and CFP (cyan) signals in the contralateral SSC (c) and the thalamus (d) of animals prepared as shown in a. Scale bar, 200 μm.(e) Quantification of the laminar distribution of CFP + /H2BYFP + starter PNs (e; L1: 0 %, L2/3: 89.9 ± 4.3 %, L4: 10.1 ± 4.3 %, L5: 0 %, L6: 0 %; 7680 cells, 3 animals) and CFP + /H2BYFP- general input neurons (f; L1: 0.2 ± 0.2 %, L2/3: 55.1 ± 2.9 %, L4: 28.7 ± 0.9 %, L5: 15.1 ± 1.9 %, L6: 1.0 ± 0.1 %; 23,317 cells, 3 animals)(g) Experimental schema for control experiments that test dependency of RV trans-synaptic spread on RG expression in starter PNs. (h) Confocal projection image merging H2BYFP (yellow) and CFP (cyan). CFP + /H2BYFP + and CFP + /H2BYFP- represent starter PNs, and general input neurons respectively of animals prepared as shown in g. Scale bar, 500 μm.(i,j) Confocal projection images with DAPI (blue) and CFP (cyan) channels in the contralateral SSC (i) and the thalamus (j) of animals prepared as shown in g. Scale bar, 200 μm.Data are presented as mean ± SEM. All experiments were repeated independently three times with similar results.

  2. Supplementary Figure. 2 Position of starter PNs from iMT within 100-μm spatial bins (Related to Figs. 37).

    (a-h) Quantification of the spatial distribution of CFP + /H2BYFP + or CFP + /HAH2B + starter PNs of individual animals for iMT with control SW animals targeting supragranular PNs (IUE at E15.5)(a), PV-INs targeting supragranular PNs (IUE at E15.5)(b), PV-INs targeting supragranular PNs (SypYFP condition)(IUE at E15.5)(c), PV-INs targeting upper supragranular PNs (IUE at E16.0)(d), PV-INs targeting granular/infragranular PNs (IUE at E12.5)(e), SOM-INs targeting aSSC supragranular PNs (IUE at E15.5)(f), SOM-INs targeting MC supragranular PNs (IUE at E15.5)(g), and VIP-INs targeting supragranular PNs (IUE at E15.5)(h). (i) Quantification of the spatial distribution of CFP + /H2BYFP + or CFP + /HAH2B + starter PNs for iMT with PV-INs targeting PNs with E16.0 (n = 5 animals), E15.5 (n = 5 animals), or E12.5 IUEs (n = 3 animals).Data are presented as mean ± SEM. See Supplementary Table 2, 3, 5, 7, 9, 11 for numerical values and statistics.

  3. Supplementary Figure. 3 RFP reporter mice are both specific and efficient. (Related to Fig. 2).

    (a) Schematic showing mouse genotype (PV-Cre;Dlx5/6-Flp;FSF-LSL-RFP) and expected recombination at dual reporter allele. (b-d) Confocal projection images of RFP (b), PV (c), and RFP/PV (d) signals in the same section from a PV-Cre;Dlx5/6-Flp;FSF-LSL-RFP mouse brain. Scale bar, 200 μm. (e) Schematic showing mouse genotype (PV-Cre;FSF-LSL-RFP) and expected recombination at dual reporter allele. (f,g) Confocal projection images taken from the same section from a PV-Cre;FSF-LSL-RFP mouse brain. RFP channel (f) and RFP/PV (g) images. Scale bar, 200 μm. (h) Specificity and efficiency of RFP expression in PV-Cre;Dlx5/6-Flp;FSF-LSL-RFP mice (n = 3 animals). (i) Schematic showing mouse genotype (FSF-LSL-RFP), genetic manipulations including IUE and RV injection, and expected recombination event at dual reporter allele. (j) Confocal projection image taken from a brain section from an FSF-LSL-RFP mouse that underwent IUE with pCAG-H2BYFP-2A-TVA-2A-RG plasmids and infection with RV-Flp-CFP viruses. H2BYFP (yellow), CFP (cyan), and RFP (red) channels are merged. Scale bar, 200 μm. (k) Schematic showing mouse genotype (FSF-RFP), genetic manipulations including IUE and RV injection, and expected recombination event at Flp reporter allele. (l) Confocal projection images taken from a brain section from an FSF-RFP mouse that underwent IUE with pCAG-H2BYFP-2A-TVA-2A-RG plasmids and infection with RV-Flp-CFP viruses. CFP (cyan) and RFP (red) channels are shown. Scale bar, 50 μm. Data are presented as mean ± SEM. All experiments were repeated independently three times with similar results.

  4. Supplementary Figure. 4 Optimization for sparse expression of HAH2B, TVA, and RG in supragranular PNs (Related to Fig. 4)

    . (a) Experimental schema for sparse expression of HAH2B, TVA, and RG in supragranular PNs. (b) Number of HAH2B + PNs in the 500 μm A-P extent of electroporated cortical domains at distinct concentrations of pCAG-DreER plasmids (0.05 μg/μL: 9.0 ± 1.0, n = 3 animals; 0.1 μg/μL: 34.0 ± 7.2, n = 3 animals; 0.25 μg/μL: 96.0 ± 0.0, n = 1 animal). (c) Histogram showing the number of HAH2B + PNs in serial 60 μm sections (0.05 μg/μL pCAG-DreER). (d) Confocal projection images taken from serial brain sections co-electroporated with pCAG-RSR-HAH2B-2A-TVA-2A-RG and pCAG-DreER (0.05 μg/μL) plasmids. DAPI (blue) and HAH2B (yellow). Arrowheads indicate HAH2B + PNs. Scale bar, 500 μm. Data are presented as mean ± SEM. All experiments were repeated independently three times with similar results.

  5. Supplementary Figure. 5 Sparse L2/3 localized starter PNs exhibit similar patterns of iMT infection and innervation by PV-INs (Related to Fig. 4).

    (a-d) iMT of input PV-INs innervating a sparsely labeled supragranular PNs. Confocal projection image of HAH2B (yellow) signal showing sparse expression of HAH2B in supragranular PNs (a) and infected CFP + starter PNs and general input neurons (b). Closed and open arrowheads represent infected CFP + /HAH2B + starter PNs and non-infected HAH2B + PNs, respectively. Merged and single-channel images from a single optical section of a CFP + /HAH2B + starter PN indicated by closed arrowhead in b (c). Confocal projection images of RFP + input PV-INs (red) that send inputs to CFP + /HAH2B + PNs shown in a-c (d). Scale bars, 100 μm (a,b,d) and c) and 10 μm (c). (e-h) Quantification of laminar distribution of CFP + /HAH2B + starter PNs (e), CFP + /HAH2B- general input neurons (f), RFP + input PV-INs (g), and RFP + processes (h) (n = 5 animals). Data are presented as mean ± SEM. All experiments were repeated independently five times with similar results. See Supplementary Table 4 for numerical values.

  6. Supplementary Figure. 6 Distance of input PV-INs from individual starter PNs (Related to Fig. 4).

    (a-f) Histograms of the frequency of RFP + PV-INs by absolute distance from their associated supragranular starter PNs for individual starter PNs 1–5 (a-e) and an aggregation of all five starter PNs (f).

  7. Supplementary Figure. 7 Colocalization of SOM and VIP in RFP+ neurons from iMT (Related to Figs. 6 and 7).

    (a-c) Confocal projection images from SOM iMT experiment showing RFP + /SOM + and RFP + /SOM- neurons indicated by closed and open arrowheads, respectively. SOM is stained with anti-SOM antibodies (green). (b) Confocal projection images from VIP iMT experiment showing RFP + /VIP + neuron indicated by closed arrowhead. VIP is stained with anti-VIP antibodies (green). (c) Confocal projection images from VIP iMT experiment showing RFP + /VIP + neuron indicated by closed arrowhead. VIP is stained using anti-VIP mRNA FISH probes (green). Scale bar, 50 μm. All experiments were repeated independently three times with similar results.

  8. Supplementary Figure. 8 Positions of starter PNs, input INs, and processes from iMT within 100-μm spatial bins (Related to Figs. 37).

    (a-f) Quantification of the spatial distribution of starter PNs (black), RFP + input INs (red), and RFP + /SypYFP + signal (magenta or green) of PV-INs targeting supragranular PNs (IUE at E15.5)(a) (n = 5 animals), PV-INs targeting supragranular PNs (SypYFP condition)(IUE at E15.5)(b) (n = 5 animals), PV-INs targeting granular/infragranular PNs (IUE at E12.5)(c) (n = 3 animals), SOM-INs targeting supragranular PNs in aSSC (IUE at E15.5)(d) (n = 5 animals), SOM-INs targeting supragranular PNs in MC (IUE at E15.5)(e) (n = 5 animals), and VIP-INs targeting supragranular PNs (IUE at E15.5)(f) (n = 5 animals). (g-h) Comparison of the quantification of the spatial distribution of RFP + input SOM-INs (g) and RFP + processes (h) between aSSC (peach) and MC (red) (IUE at E15.5) (n = 5 animals each). Data are presented as mean ± SEM. See Supplementary Table 5, 7, 9, 11 for numerical values.

  9. Supplementary Figure. 9 Similar axonal projections to L1 by total SOM-INs in the aSSC and the MC (Related to Fig. 6).

    (a,b) Confocal projection images of RFP + SOM-INs (red) in the aSSC (a) and the MC (b) of a Cre-dependent RFP reporter mouse with SOM-Cre allele. Upper panels show the laminar distribution of somata and axons of all SOM-INs. Lower panels highlight L1 axons from SOM-INs. Scale bars, 200 μm (upper panels), 25 μm (lower panels). (c) Area occupied by L1 RFP + processes normalized to the number of RFP + somata in the MC and the aSSC (n = 3 animals each). Data are presented as mean ± SEM. All experiments were repeated independently three times with similar results. See Supplementary Table 3 for numerical values and statistics.

  10. Supplementary Figure. 10 Summary cartoon of iMT results.

    (a) Summary of organization of input PV-INs that innervate supragranular or infragranular PNs in the SSC. (b) Summary of organization of input SOM-INs that innervate supragranular PNs in the aSSC or the MC. (c) Summary of organization of input VIP-INs that innervate supragranular PNs in the SSC.

Supplementary information

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Further reading

Fig. 1: iMT of cortical IN subtypes that send direct inputs to defined PNs.
Fig. 2: iMT specifically labels PV-INs sending direct inputs to supragranular PNs.
Fig. 3: Supragranular PNs receive not only local but also translaminar inputs from PV-INs.
Fig. 4: A single supragranular PN in the SSC receives inputs from PV-INs in multiple layers.
Fig. 5: Granular and infragranular PNs receive local inputs from granular and infragranular PV-INs.
Fig. 6: Unique cellular and axonal organization of SOM-INs that innervate supragranular PNs in distinct cortical areas.
Fig. 7: Cellular and axonal organization of VIP-INs that innervate supragranular PNs in the SSC.
Supplementary Figure. 1: Mono-trans-synaptic spread of RV-Flp-CFP viruses depends on expression of RG in starter PNs (Related to Figs. 1 and 2).
Supplementary Figure. 2: Position of starter PNs from iMT within 100-μm spatial bins (Related to Figs. 37).
Supplementary Figure. 3: RFP reporter mice are both specific and efficient. (Related to Fig. 2).
Supplementary Figure. 4: Optimization for sparse expression of HAH2B, TVA, and RG in supragranular PNs (Related to Fig. 4)
Supplementary Figure. 5: Sparse L2/3 localized starter PNs exhibit similar patterns of iMT infection and innervation by PV-INs (Related to Fig. 4).
Supplementary Figure. 6: Distance of input PV-INs from individual starter PNs (Related to Fig. 4).
Supplementary Figure. 7: Colocalization of SOM and VIP in RFP+ neurons from iMT (Related to Figs. 6 and 7).
Supplementary Figure. 8: Positions of starter PNs, input INs, and processes from iMT within 100-μm spatial bins (Related to Figs. 37).
Supplementary Figure. 9: Similar axonal projections to L1 by total SOM-INs in the aSSC and the MC (Related to Fig. 6).
Supplementary Figure. 10: Summary cartoon of iMT results.